Bacteria occupy countless niches. They respond to innumerable environmental situations to ensure their survival and to allow them to become pathogenic. In times of change/hostility bacteria encode genes normally unexpressed. Certainly, there is not a single type of response - each situation requires a specific strategy. Bacteria sense signals in the environment, recognize its composition and initiate the correct survival approach. With this in mind, and using a variety of techniques including NMR, mass spectrometry, mutagenesis and chemical synthesis, our work focuses in three areas: (i) transition state regulator proteins (TSRs); (ii) two-component signaling proteins; and (iii) small molecules that overcome the bacterial stress response, including biofilm formation. The transition state is a cellular holding pattern during which bacteria decide which protective strategy is most appropriate. Incorrect signaling during this time results in cell death. TSRs control numerous pathways by their ability to bind to a diverse array of gene promoter regions which have no consensus DNA sequence. The mechanism by which they target their DNA targets is novel and very different than the classical consensus sequence recognition model. We will perform detailed comparative studies on three structurally homologous TSRs (AbrB, Abh and SpoVT) to better understand how DNA targets are recognized by this new class of proteins. We will also study the newly discovered protein AbbA which may influence the transition state by a completely novel mechanism. When the cell decides which response is necessary in order to survive, ubiquitous signaling modules known as the two-component system/phosphorelay are called upon. These modules are responsible for the majority of all sensory-response functions in bacteria. Understanding their mechanism of action is essential since they are significant targets for antimicrobial therapeutics. We will provide the very first characterization of a two-domain response regulator in solution (Spo0A) and elucidate the structure, dynamics, interactions and recognition determinants of multiple proteins from a complex phosphorelay that controls biofilm development in all human pathogenic Vibrio species. Finally, we have recently discovered a class of synthetically accessible, non-toxic small molecules that act as anti-biofilm agents with unprecedented effectiveness. We have recently discovered that these compounds also completely re-sensitize bacteria to conventional antibiotics and have demonstrated their effectiveness against multi-drug resistant bacterial strains including MRSA. We will develop further analogues of our compounds and will detail their effects, as adjuvant therapies, on the performance of many current antibiotics against multiple bacterial strains. We will also develop new toxicology screens and initiate differential array work to begin determining the mode of action of our compounds. Our overall goals are: (a) to better understand how bacteria are able to adapt and reach their pathogenic potential and (b) develop approaches to stop pathogenic bacteria becoming infectious. PUBLIC HEALTH RELEVANCE: Bacteria survive and reach their pathogenic potential towards humans by their ability to invoke the correct protective strategies at the appropriate times. In order to better define anti-microbial therapeutic targets, we will elucidate the mechanism of action for protein signaling cascades involved in the transition state and in biofilm formation. In addition we will further develop powerful new, non-toxic, small molecules that have been shown to both eradicate insidious pathogenic biofilms and to re-sensitize resistant bacteria to conventional antibiotics.